Characterization of Nef vesicles and their effect on T cells and macrophage

描述(申请人提供)：感染人类免疫缺陷病毒1型(HIV-1)会导致免疫系统的失调和最终的免疫系统衰竭，称为获得性免疫缺陷综合症(AIDS)。CD4T细胞逐渐耗尽，感染者最终死于各种机会性感染。HIV-1是如何导致艾滋病的确切机制还不是很清楚。很明显，感染HIV-1会导致CD4T细胞耗尽，免疫系统处于高度激活状态。然而，这些过程的确切机制仍然难以捉摸。在这一应用中，我们建议探索病毒蛋白Nef在艾滋病发病机制中可能发挥的作用。我们的初步工作表明，Nef是由HIV感染的细胞以外周体样小泡的形式分泌的。NEF小泡存在于感染者的血浆中，我们的数据表明Nef小泡的水平与抗逆转录病毒治疗后CD4T细胞的恢复有关。我们推测，感染细胞以小泡的形式分泌的Nef可以诱导T细胞和巨噬细胞的作用，并至少导致艾滋病的部分发病机制。在这一应用中，我们建议研究泡状Nef及其与原始T细胞和巨噬细胞的相互作用。Nef囊泡的组成将通过磁珠分离以及结合囊泡的流式细胞仪和蛋白质组学鉴定相结合的方法来确定。从感染者血浆中分离出的NEF囊泡将用于治疗原始的CD4和CD8T细胞和巨噬细胞。将使用96孔阵列对处理后的细胞进行分析，以确定诱导凋亡、免疫激活、细胞因子表达和基因表达。收集到的信息将使我们更好地了解分泌型Nef在艾滋病发病机制中的作用。这些信息可能导致针对分泌的Nef的分泌或作用的新的治疗方法。 公共卫生相关性：艾滋病毒-艾滋病是一种以破坏正常免疫力为特征的疾病。在本申请中，我们提出，感染艾滋病毒会导致大量类似病毒的信息包释放，这种信息包被称为“外显体”。我们的理论是，外切体的释放至少是艾滋病患者免疫功能丧失的部分原因。

DESCRIPTION (provided by applicant): Infection with Human Immunodeficiency Virus type 1 (HIV-1) results in dysregulation and the ultimate depletion of the immune system known as Acquired Immunodeficiency Syndrome (AIDS). CD4+ T cells are gradually depleted and the infected individuals eventually succumb to a variety of opportunistic infections. The exact mechanisms of how HIV-1 induces AIDS are not well understood. It is clear that infection with HIV-1 results in depletion of CD4+ T cells and a state of hyperactivation of the immune system. However, the exact mechanism of these processes remains elusive. In this application, we propose to explore what role the viral protein Nef might play in AIDS pathogenesis. Our preliminary work shows that Nef is secreted from HIV infected cells in the form of exosome-like vesicles. Nef vesicles are present in the plasma of infected individuals and our data suggests Nef vesicle levels correlate with recovery of CD4+ T cells after antiretroviral therapy. We hypothesize that Nef, secreted from infected cells in the form of vesicles can induce effects on T cells and macrophage and cause at least some of the pathogenesis observed in AIDS. In this application we propose to study vesicular Nef and its interaction with primary T cells and macrophage. The composition of Nef vesicles will be determined using a combination of magnetic bead separation followed by characterization of bound vesicles by flow cytometry and Proteomics. Nef vesicles isolated from the plasma of infected individuals will be used to treat primary CD4 + and CD8 + T cells and macrophage. The treated cells will be analyzed to determine induction of apoptosis, immune activation, cytokine expression and gene expression using 96-well arrays. Together the information gathered will give us a better picture of the role of secreted Nef in the pathogenesis of AIDS. Such information could lead to new therapeutic approaches that target secretion or the action of secreted Nef. PUBLIC HEALTH RELEVANCE: HIV-AIDS is disease characterized by the destruction of normal immunity. In this application we propose that infection with HIV causes the release of large numbers of virus-like packets called "exosomes". Our theory is that release of exosomes is responsible for at least some of the loss of immune function seen in AIDS.